Q.34 An oligonucleotide was sequenced by the dideoxy method of Sanger and the following
autoradiogram was obtained
The sequence of the oligonucleotide is
(A) 3’-GTCCTGTACA-5’
(B) 5’-GTCCTGTACA-3’
(C) 5’-ACATGTCCTG-3’
(D) 3’-AATTTCCCGG-5’
Introduction
The Sanger dideoxy sequencing method is a classical DNA sequencing technique widely used in molecular biology.
Competitive exams such as CSIR-NET, GATE, and DBT often test the ability to interpret an autoradiogram
obtained from this method. This article explains how to read the sequencing gel and identify the correct oligonucleotide sequence.
Principle of Sanger Sequencing
- Four separate reactions contain ddATP, ddTTP, ddCTP, and ddGTP.
- Incorporation of a dideoxynucleotide terminates DNA synthesis.
- Fragments are separated by size using polyacrylamide gel electrophoresis.
- Smaller fragments migrate faster and appear at the bottom of the gel.
How to Read the Autoradiogram
- Read the bands from bottom to top.
- The sequence is always written in the 5′ → 3′ direction.
- Each lane (A, T, C, G) corresponds to the terminating base.
Sequence Deduction
Reading the autoradiogram from bottom to top across the lanes gives the following base order:
Final Sequence:
5′-GTCCTGTACA-3′
Correct Answer
Option (B): 5′-GTCCTGTACA-3′
Conclusion
Correct interpretation of a Sanger sequencing autoradiogram requires careful attention to
band order and strand direction. By reading the gel from bottom to top and writing the sequence
in the 5′ → 3′ direction, the correct oligonucleotide sequence is easily determined.
